DNA Microarray Protocol


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DNA Microarray Vendors and Products

Basic DNA Microarray Protocol:

The Stages in a Microarray Experiment:

1. Obtain DNA Microarray Chip

Currently, over 50,000 cDNA probes can be spotted onto a 25X75mm slide by robotic printing. It is thus possible to array the entire human genome compliment using this high-density array approach (e.g. Affymetrix Arrays).

Thousands of distinct spots of known oligos or cDNAs are printed on matrix platforms located on either silicon, nylon sheets, or glass slides.

2. Cy3 and Cy5 Labeling of RNA Specimens using (RT-PCR) cDNA synthesis

The RNA from 2 or more specimens are compared. The RNA from each specimen is reverse transcribed using RT-PCR to cDNA (complementary cDNA). The cDNA is labeled with two different fluorescent dyes which are colored differently (red and green), with Cy3 and Cy5.

3. Hybridization of cDNA Probes to DNA array

The cDNA probes are hybridized to the DNA microarray. The DNA array is then washed.

4. Detection of Hybridization Signal

Fluorescent hybridization signals are scanned and detected using laser confocal devices. Data is the filtered and processed.

5. DNA Microarray Data Analysis

Data analysis is conducted using DNA Microarray bioinformatic tools and/or image processing software.

The signal intesities of the spots are correlated with the concentrations of target mRNA samples. Data mining is also conducted using statistical programs and algorithms to determine if the gene of interest is up-regulated, down-regulated, or unchanged.

Data is then organized using a database. Gene Annotation can be then performed on the data, with GO (gene ontology) analysis, clustering analysis (which groups similar genes and pathways together), and analysis of pathways and networks of genes.

Microarray Types and Info

Using Microarrays

DNA Microarray Protocol